Fibrinogen is one of the numerous proteins of blood plasma from which the phenomena and mechanisms emanate to form the structure of the fibrin clot. Its ubiquitous physiological role in internal restructuring or repair of tissue discontinuity has been extended to a corresponding role of external application developed over the past scores of years as a concentrate processed from plasma for tissue bonding under such descriptive terms as fibrin glue, fibrin adhesive, fibrin weld, fibrin sealant, and so on.
The clinical use of fibrin prepared from plasma by various methods of cryoprecipitation and chemical insolubilization has gradually emerged for such early uses as a hemostyptic adhesive powder with small open vessels (Berger, 1909), as a hemostatic agent in cerebral surgery (Grey, 1915), in suturing of peripheral nerves (Matras et al, 1972), and gradually expanding to the repair of traumatized tissues (Brands et al, 1982), and the anastomoses or restructuring of cardiovascular, colon, bronchial sections, severed nerve endings, and other anatomical discontinuities currently in wide-spread practice often replacing or augmenting conventional suturing. In such clinical applications, the native fibrinogen content in human plasma averages 513 milligrams per deciliter (mg/dcl) according to standard clinical assays, and ranges from 229 mg/dcl to 742 mg/dcl standard deviation, based on photometric measurements of turbidity from clotting (Castillo, 1989). This range of native concentration, corresponding to 0.229% to 0.742% (averaging 0.531%) of the human population, is too dilute and too fluid with the aqueous burden being principally water, for direct application to tissue site for direct or assisted bonding.
The separation and/or concentration of fibrinogen emanated from precipitation using concentrated salt solutions, such as semi-saturated sodium chloride (Hamerstein, 1878) and saturated ammonium sulfate, cold (-3.degree. C.) ethanol (Cohn et al. 1946), other low molecular weight organic liquids (Pennell, 1960) and amino acids (Edsall and Lever, 1951), and numerous combinations of these precedent precipitations. These procedures, using excessive osmolal additives based on physicochemical gradations of solubility, were applied to the preparation of high purity, single fibrinogen protein entities stripped of the natively associated proteins, notably glycoproteins and other peptides, for analytical and molecular characterization.
The clinical preparations of cryoprecipitation at the -80.degree. C. range have been applied from at least 6 hours (Gestring, 1982) to the general practice of 24 hours (Dresdale et al, 1985; Spotnitz et al, 1987); in another instance, plasma was "deep frozen" at -18.degree. C. for 72 hours presumably with a longer time to compensate for the difference to the lower cryogenic precipitation. In none of these general practices has there been any indication of the concentrate yield or product characterization and quality. All of these preparations require prolonged preparation times.
The thawing time in numerous known, published methods has not been consistent and in no instances related to either the quantity or quality of the attained fibrinogen concentrates. For instance, the thawing time may vary from such indefinite temperature-time kinetics as at 4.degree. C. "when liquid" (Gestring, 1982), for "several hours" at 4.degree. C. (Dresdale, 1985a), after slow rethawing at 4.degree. C. (Dresdale, 1985b), slowly thawed at 1.degree. C. to 60.degree. C. for 20 hours (Siedentop, 1985), and also at 1.degree. C. to 60.degree. C. for 12 hours; in no instance has there been any indication of yield, solids content, or clinical assay of the fibrinogen content. In all these instances prolonged thawing with re-dissolving of the cryoprecipitates during the temperature-time thermal drift, lead to inordinate loss of fibrinogen and its associated proteins with solids contents varying from as little as 3% to 6%; the factor (.times.) of concentrating efficiency based on initial 0.531% fibrinogen amounts to only 5.9.times. to 11.7.times..
The current practices of centrifugation involve a wide range of speed (RPM), gravitational force (.times.g), temperatures, and time. These include, for instance, unspecified cold centrifuge at 2300 g for 10 to 15 minutes (Gestring, 1982), thawed cryoprecipitate at 1000.times. g for 15 minutes (Dresdale 1985), 1.degree. C. to 6.degree. C. at 5,000 rpm (unspecified .times.g) for 5 minutes (Siedentop, 1985), room ambient temperature at 10.000.times. g for 20 minutes (Epstein, 1986), and 4.degree. C. at 6500.times. g for 5 minutes (Spotnitz, 1987). The last named is the only source that provided a yield averaging 11.7 ml of fibrinogen concentrate from 250 cc plasma with a calculated solids content of 3.9%.
The present state of the art in applying reproducible engineering processing components as a system of integrated temperature time kinetics is not only confusing and completely devoid of corresponding overall temperature time force controls but also completely lacking in relating the kinetics to process efficiency in terms of the yields and assay of the derived fibrinogen protein concentrates.
Therefore, a process for preparing fibrinogen concentrates having a higher level of solids content with specified and defined specifications is needed for the ever increasing clinical demand for fibrinogen and related clottable factors (Epstein et al., 1990). Furthermore, this need is cogent not only for large scale production from pooled blood fractions but particularly for small lot ready preparation of fibrinogen concentrate from autologous patient plasma in view of the prevalent risks of viral infection, notably numerous forms of hepatitis and particularly human immunodeficiency virus, from pooled or single donor sources. In addition, small packaged lots may be prepared by the process of the present invention for use in emergencies.